Lymph nodes are the initial sites of metastasis for most solid tumors, including breast carcinoma and melanoma. Progression from lymph node metastases to distant metastases is suggested by the clinical record as excision of tumor-bearing, draining lymph nodes can improve clinical outcome for many tumor types. Surprisingly, little is known about the factors in the lymph node microenvironment that may contribute to tumor metastasis. Our preliminary data show that primary tumors precondition draining as well as distal lymph nodes for tumor metastasis by inducing integrin ?4?1 and VEGF-R3-mediated lymphangiogenesis but not hemangiogenesis in lymph nodes. Lymph node lymphangiogenesis is then required for tumor metastasis to lymph nodes. Selective inhibition of integrin ?4?1 or VEGF-R3 in lymph nodes blocks lymph node lymphangiogenesis and prevents tumor metastasis to lymph nodes. In contrast, stimulation of lymph node lymphangiogenesis by injections of VEGF-C accelerates metastasis to stimulated lymph nodes. Stimulation of lymph node lymphangiogenesis in normal mice also promoted tumor cell homing to and extravasation into stimulated lymph nodes even in the absence of a primary tumor when tumor cells were injected directly into the lymphatic system. This homing could be blocked by suppressing lymphangiogenesis with antagonists of integrin ?4?1 or VEGF-R3. These studies suggest that lymph node lymphangiogenesis directly promotes lymph node metastasis by enhancing tumor cell adhesion and extravasation into the node. To further our understanding of the role of the lymph node microenvironment in tumor metastasis, we will test the hypotheses that integrin ?4?1 mediated signaling via paxillin promotes lymphatic endothelial cell migration and that integrin ?4?1 mediated signaling via Protein Kinase A, Raf and caspase 8 promote lymphatic endothelial cell survival during lymphangiogenesis and subsequent tumor metastasis; that lymph node lymphatic endothelium promotes the homing, adhesion and extravasation of tumor cells by expressing key tumor cell chemoattractants such as CCL21 as well as tumor cell adhesion ligands/receptors, such as integrin ?4?1, VCAM and E-Selectin; and that inflammatory mediators and bone marrow derived cells within the lymph node play roles in promoting tumor metastasis. The aims of this proposal are: 1) To determine the role of integrin ?4?1-paxillin interaction during lymphatic endothelial cell migration; 2) To determine the roles of protein kinase A, Raf and caspase 8 in integrin ?4?1 mediated lymphatic endothelial cell survival during lymphangiogenesis; 3) To evaluate the roles of specific chemokine and adhesion receptors in promoting tumor cell attraction and adhesion to lymphatic endothelium during tumor metastases; 4) To determine the contributions of inflammatory mediators and cells in lymph node lymphangiogenesis and metastasis. Importantly, our preliminary findings on the role of lymph node lymphangiogenesis in tumor metastasis indicate that detection of lymph node lymphangiogenesis may facilitate cancer diagnosis by helping to predict the sites of metastatic lesions yet to form. These results also suggest that inhibitors of lymph node lymphangiogenesis may play important roles in cancer therapeutics. The studies proposed in this application are likely to lead to the development and testing of new agents to diagnose and prevent or treat metastatic cancer.